RESUMO
Cape gooseberry (Physalis peruviana) is an exotic fruit highly valued, however it is a very rich source of polyphenol oxidase (PPO). In this study, Cape gooseberry PPO was isolated and biochemically characterized. The enzyme was extracted and purified using acetone and aqueous two-phase systems. The data indicated that PPO had the highest substrate affinity for chlorogenic acid, 4-methylcatechol and catechol. Chlorogenic acid was the most suitable substrate (Km=0.56±0.07 mM and Vmax=53.15±2.03 UPPO mL(-1) min(-1)). The optimal pH values were 5.5 for catechol and 4-methylcatechol and 5.0 for chlorogenic acid. Optimal temperatures were 40°C for catechol, 25°C for 4-methylcatechol and 20°C for chlorogenic acid. In inhibition tests, the most potent inhibitor was found to be ascorbic acid followed by L-cysteine and quercetin. This study shows possible treatments that can be implemented during the processing of Cape gooseberry fruits to prevent browning.
Assuntos
Catecol Oxidase/isolamento & purificação , Frutas/enzimologia , Physalis/enzimologia , Ácido Ascórbico/análise , Catecol Oxidase/antagonistas & inibidores , Catecóis/química , Ácido Clorogênico/química , Cisteína/análise , Estabilidade Enzimática , Peso Molecular , Especificidade por Substrato , TemperaturaRESUMO
Residual enzymatic activity in certain foods, particularly of polyphenoloxidase (PPO), is responsible for the majority of anthocyanin degradation in food systems, causing also parallel losses of other relevant nutrients. The present work explored the feasibility of modifying phenolic profiles of thyme extracts, by use of chromatographic resins, to obtain phenolic extracts capable of enhancing anthocyanin colour and stability in the presence of PPO activity. Results indicated that pretreatment of thyme extracts with strong-anion exchange resins (SAE) enhanced their copigmentation abilities with strawberry juice anthocyanins. Phenolic chromatographic profiles, by HPLC-PDA, also demonstrated that thyme extracts subjected to SAE treatments had significantly lower concentrations of certain phenolic compounds, but extracts retained their colour enhancing and anthocyanin stabilization capacities though copigmentation. Additional testing also indicated that SAE modified extract had a lower ability (73% decrease) to serve as PPO substrate, when compared to the unmodified extract. Phenolic profile modification process, reported herein, could be potentially used to manufacture modified anthocyanin-copigmentation food and cosmetic additives for colour-stabilizing applications with lower secondary degradation reactions in matrixes that contain PPO activity.
Assuntos
Antocianinas/química , Antioxidantes/química , Bebidas/análise , Catecol Oxidase/antagonistas & inibidores , Fragaria/química , Thymus (Planta)/química , Resinas de Troca Aniônica/química , Antioxidantes/isolamento & purificação , Cor , Conservação de Alimentos/métodos , Humanos , Oxirredução , Fenóis/química , Extratos Vegetais/químicaRESUMO
Polyphenol oxidase (PPO) is an enzyme widely distributed in the plant kingdom that has been detected in most fruits and vegetables. PPO was extracted and purified from Manila mango (Mangifera indica), and its biochemical properties were studied. PPO was purified 216-fold by hydrophobic interaction and ion exchange chromatography. PPO was purified to homogeneity, and the estimated PPO molecular weight (MW) by SDS-PAGE was ≈31.5 kDa. However, a MW of 65 kDa was determined by gel filtration, indicating a dimeric structure for the native PPO. The isolated PPO showed the highest affinity to pyrogallol (Km = 2.77 mM) followed by 4-methylcatechol (Km = 3.14 mM) and catechol (Km = 15.14 mM). The optimum pH for activity was 6.0. PPO was stable in the temperature range of 20-70 °C. PPO activity was completely inhibited by tropolone, ascorbic acid, sodium metabisulfite, and kojic acid at 0.1 mM.
Assuntos
Catecol Oxidase/isolamento & purificação , Catecol Oxidase/metabolismo , Mangifera/enzimologia , Catecol Oxidase/antagonistas & inibidores , Catecóis/metabolismo , Cromatografia em Gel , Cromatografia por Troca Iônica , Inibidores Enzimáticos/farmacologia , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Peso Molecular , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/metabolismo , Pirogalol/metabolismo , Especificidade por Substrato , TemperaturaRESUMO
The Royal Sun mushroom, the Himematsutake culinary-medicinal mushroom, Agaricus brasiliensis has several polyphenoloxidase activities in a broad sense. Here we report the partial purification of tyrosinase-type polyphenoloxidase (PPO). PPO is purified from A. brasiliensis without browning using a two-phase partitioning with Triton X-114 and ammonium sulfate fractionation. Partially denaturing SDS-PAGE (sodium dodecyl sulfate-polyacrylamide electrophoresis) staining with L-3,4-dihydroxyphenylalanine was performed and the indicated molecular sizes were approximately 70 kDa and 45 kDa. The purified enzyme is in its latent state and can be activated maximally in the presence of 1.6 mM sodium dodecyl sulfate (SDS). This enzyme catalyzes two distinct reactions, monophenolase and diphenolase activity, and the monophenolase activity showed a lag time typical of polyphenoloxidase. The K(m) value for 4-tert-butylcatechol was quite similar in the presence and absence of SDS, but the apparent V(max) value was increased 2.0-fold by SDS. Mimosine was a typical competitive inhibitor with K(i) values of 138.2 microM and 281.0 microM n the presence and absence of SDS, respectively.
Assuntos
Agaricus/enzimologia , Catecol Oxidase/metabolismo , Carpóforos/enzimologia , Catecol Oxidase/antagonistas & inibidores , Catecol Oxidase/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática/efeitos dos fármacos , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Monofenol Mono-Oxigenase/isolamento & purificação , Monofenol Mono-Oxigenase/metabolismo , Dodecilsulfato de Sódio/farmacologia , TemperaturaRESUMO
While a long shelf life for fruit products is highly desired, enzymatic browning is the main cause of quality loss in fruits and is therefore a main problem for the food industry. In this study polyphenol oxidase (PPO), the main enzyme responsible for browning was isolated from mamey fruit (Pouteria sapota) and characterized biochemically. Two isoenzymes (PPO 1 and PPO 2) were obtained upon ammonium sulfate precipitation and hydrophobic and ion exchange chromatography; PPO 1 was purified up to 6.6-fold with 0.28% yield, while PPO 2 could not be characterized as enzyme activity was completely lost after 24 h of storage. PPO 1 molecular weight was estimated to be 16.1 and 18 kDa by gel filtration and SDS-PAGE, respectively, indicating that the native state of the PPO 1 is a monomer. The optimum pH for PPO 1 activity was 7. The PPO 1 was determined to be maximum thermally stable up to 35°C. Kinetic constants for PPO 1 were K(m)=44 mM and K(m)=1.3 mM using catechol and pyrogallol as substrate, respectively. The best substrates for PPO 1 were pyrogallol, 4-methylcatechol and catechol, while ascorbic acid and sodium metabisulfite were the most effective inhibitors.
Assuntos
Catecol Oxidase/metabolismo , Frutas/enzimologia , Pouteria/enzimologia , Ácido Ascórbico/farmacologia , Catecol Oxidase/antagonistas & inibidores , Catecol Oxidase/isolamento & purificação , Catecóis/metabolismo , Cromatografia por Troca Iônica , Eletroforese em Gel de Poliacrilamida , Concentração de Íons de Hidrogênio , Isoenzimas/isolamento & purificação , Isoenzimas/metabolismo , México , Peso Molecular , Pirogalol/metabolismo , Sulfitos/farmacologia , TermodinâmicaRESUMO
Apple peel is a waste product from dried apple manufacture. The content of phenolic compounds, dietary fiber, and mineral are higher in apple peel, compared to other edible parts of this fruits. The objective of this study was to develop an ingredient from Granny Smith apple peel, using a pilot scale double drum-dryer, as drying technology. The control of all steps to maximize the retention of phenolic compounds and dietary fiber was considered. Operational conditions, such as drying temperature and time were determined, as well as important preprocessing steps like grinding and PPO inhibition. In addition, the physical-chemical characteristics, mineral and sugar content, and technological functional properties such as water retention capacity, solubility index, and dispersability among others, were analyzed. A simple, economical, and suitable pilot scale process, to produce a powder ingredient from apple peel by-product, was obtained. The drying process includes the application of ascorbic acid at 0.5% in the fresh apple peel slurry, drum-dryer operational conditions were 110 degrees C, 0.15 rpm and 0.2 mm drum clearance. The ingredient developed could be considered as a source of phenolic compounds (38.6 mg gallic acid equivalent/g dry base) and dietary fiber (39.7% dry base) in the formulation of foods. Practical Application: A method to develop an ingredient from Granny Smith apple peel using a pilot scale double drum-dryer as drying technology was developed. The method is simple, economical, feasible, and suitable and maximizes the retention of phenolic compounds and dietary fiber present in the raw matter. The ingredient could be used in the formulation of foods.
Assuntos
Fibras na Dieta/análise , Flavonoides/análise , Alimentos Fortificados , Frutas/química , Malus/química , Fenóis/análise , Antioxidantes/administração & dosagem , Ácido Ascórbico/química , Catecol Oxidase/antagonistas & inibidores , Catecol Oxidase/metabolismo , Fenômenos Químicos , Fibras na Dieta/administração & dosagem , Inibidores Enzimáticos/química , Flavonoides/administração & dosagem , Manipulação de Alimentos/instrumentação , Manipulação de Alimentos/métodos , Indústria de Processamento de Alimentos/economia , Temperatura Alta , Concentração de Íons de Hidrogênio , Resíduos Industriais/análise , Resíduos Industriais/economia , Fenóis/administração & dosagem , Proteínas de Plantas/antagonistas & inibidores , Proteínas de Plantas/metabolismo , Polifenóis , Solubilidade , Fatores de Tempo , Oligoelementos/administração & dosagem , Oligoelementos/análise , Água/análiseRESUMO
Polyphenol oxidase (PPO) was isolated from the B-serum obtained after repetitive freeze-thawing of the bottom fraction isolated from ultracentrifuged fresh latex. The B-serum was subjected to acetone precipitation and CM-Sepharose chromatography, affording two PPOs, PPO-I and PPO-II, which, upon SDS-PAGE, were 32 and 34 kDa, respectively. Both PPOs possessed the same pI (9.2), optimum pH (7) and optimum temperature (35-45 degrees C). They are stable up to 60 degrees C and active at broad pH ranges from 4-9. The K(m) values of PPO-I for dopamine, L-dopa and catechol as substrates are 2.08, 8.33 and 9.09 mM, while those for PPO-II are 2.12, 4.76 and 7.14 mM, respectively. Among various PPO inhibitors tested, 4-hexylresorcinol was the most potent. Anionic detergents were among the most effective activators of the enzymes, while cationic and nonionic detergents showed little and no effect on the PPO activities, respectively.
Assuntos
Catecol Oxidase/isolamento & purificação , Catecol Oxidase/metabolismo , Hevea/química , Látex/química , Catecol Oxidase/antagonistas & inibidores , Catecol Oxidase/química , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Concentração de Íons de Hidrogênio , Cinética , Peso Molecular , Especificidade por Substrato , TemperaturaRESUMO
Prophenoloxidase (proPO) was purified from blood cells of the brown shrimp Penaeus californiensis by ultracentrifugation and dye affinity chromatography. The isolated proPO is a 114-kDa monomeric protein as determined by SDS-PAGE. This protein can be hydrolyzed by proteinases, producing a 107-kDa active phenoloxidase (PO). The isoelectric point for both protein forms was 7.35. The PO reaction using L-DOPA as substrate, has an optimum pH of 8, and was poorly inhibited by sodium azide, thiourea and EDTA, but strongly inhibited by diethyl thiocarbamate. According to the substrate affinity and inhibition characteristics, this phenoloxidase was classified as a tyrosinase-like phenoloxidase. Purified proPO was not activated by bacterial lipopolysaccharides or beta-glucans.
Assuntos
Catecol Oxidase/sangue , Precursores Enzimáticos/sangue , Hemócitos/enzimologia , Penaeidae/enzimologia , Animais , Catecol Oxidase/antagonistas & inibidores , Catecol Oxidase/isolamento & purificação , Centrifugação , Cromatografia de Afinidade , Eletroforese em Gel de Poliacrilamida , Precursores Enzimáticos/antagonistas & inibidores , Precursores Enzimáticos/isolamento & purificação , Concentração de Íons de Hidrogênio , CinéticaRESUMO
Objective of this research was to find alternative methods for the control of polyphenol oxidase (PPO) activity in fruits and vegetables with the purpose of reducing or eliminating the use of SO2 for this purpose. Interactions between the use of ascorbic acid, citric acid, EDTA, sodium metabisulphite and heat treatment (70 degrees C for 2 min) in the control of PPO activity were studied in avocado (var. Fortuna), banana (var. Nanica), apple (var. Ana, Fuji, Gala & Golden), pear (var. D'Agua), peach (var. Réal), potato (var. Bintje), eggplant (var. Super F100), mushroom (Agaricus bisporus) and hearts-of-palm (Euterpe edulis Mart). The results demonstrated that PPO of avocado and eggplant was most resistant to inhibition by the methods used. The least efficient method tested for the control of PPO was the addition of ascorbic acid and EDTA, while the most efficient methods investigated included the use of ascorbic acid, citric acid, sodium metabisulphite and heat treatment. The results indicated that, with the exception of PPO from avocado, the most adequate alternative method to substitute for the use of SO2 in the control of PPO was a combination of ascorbic acid, citric acid and heat treatment.